UC Davis

Carbohydrates are structurally complex and functionally important biomolecules. They are presented in the forms of monosaccharides, oligosaccharides, polysaccharides, and as glycans attached to lipids and proteins forming glycoconjugates. Carbohydrates are attractive synthetic targets but very challenging to be produced in structure-defined and size-controlled manner. Glycosyltransferase-catalyzed glycosylation and one-pot multienzyme (OPME) systems are powerful tools for producing complex glycans and glycoconjugates. I have been working on characterizing and improving two bacterial sialyltransferases and developing strategies for synthesizing glycosyltransferase-dependent chemoenzymatic synthesis of complex carbohydrates with a focus on human milk oligosaccharides containing a linear hexaose core with fucose and/or sialic acid modifications. Chapter 1 introduces sialic acids, sialyltransferases, strategies and applications of OPME systems in sugar nucleotide formation, glycosyltransferase-based glycosylation, sequential OPME glycosylation, and regioselective enzymatic glycosylation by chemical and enzymatic protections. Chapter 2 describes protein engineering of two bacterial sialyltransferases and their applications in the synthesis of glycans, glycolipids, and glycoproteins. Chapter 3 focuses on the construction of a comprehensive library of para-hexaose-core-containing human milk oligosaccharides (HMOs) by a highly efficient chemoenzymatic synthetic platform guided by Enzyme Assembly Synthetic Maps (EASyMaps). Sialic acids (Sia) are monosaccharides commonly presented at the termini of carbohydrate moieties on cell surface glycoconjugates. Sialic acids with or without additional modifications are directly involved in many molecular recognition events including immune regulation, cell-cell interaction, inflammation, cancer metastasis, bacterial and viral infection. Sialyltransferases are key enzymes used by nature for catalyzing the formation of sialyl glycosidic bonds in sialic acid-containing oligosaccharides or glycoconjugates. In my doctoral research, an α2-3-sialyltransferase from Campylobacter jejuni (CjCst-I) was cloned using a synthetic gene as a template for polymerase chain reaction (PCR). With a C-terminal truncation and addition of an N-terminal maltose-binding protein (MBP) fusion tag, soluble expression of the resulting MBP-CjCst-IΔ145-His6 was increased significantly. The enzyme was applied in the synthesis of glycans, glycoproteins, and glycolipids. In addition to this enzyme, an α2-3/8-sialyltransferase CjCst-II was studied for the purpose of synthesizing unnatural sialosides with modified sialic acids. Four mutants were obtained based on protein sequence alignment and crystal structural analysis. Among those mutants, CjCst-IIΔ32-His6 I53S/N51D stood out for its improved activity towards CMP-Neu5Ac8N3 as the modified donor substrate compared to CjCst-IIΔ32-His6 I53S single mutant which was previously cloned and commonly used in sialoside synthesis in our group. This mutant will be biochemically characterized and used for the synthesis of disialyl glycosides containing a terminal 8-N-modified sialic acids in the future. Besides sialyltransferase engineering, I have worked on systematic construction of human milk oligosaccharides (HMOs), which are important glycan targets drawing interests for their synthesis and their applications as food additives and nutraceuticals. There is an increasing attention on the contribution of HMOs to the health of breast-fed infants, as they are a major component of human milk. They are not digested by infants but can serve as: prebiotics to support the growth and colonization of probiotics and suppress the growth of some pathogenic bacteria, anti-adhesive decoy receptors for some pathogenic microbes, antibiofilm antimicrobials, brain-gut axis modulators, immune modulators, infant colon epithelial cell response modulators, and cell maturation stimulators. Detailed functions of specific HMOs, especially those with more complex structures, are not clear. Exploring applications of HMOs as infant formula additives, nutraceuticals, and/or therapeutics has begun but has been slow due to the limited access to structurally defined HMOs in sufficient amounts. Chapter 3 describes the development of a highly efficient user-friendly glycosyltransferase-based synthetic platform to access target HMOs in a systematic manner with a focus on HMOs containing a linear hexaose core, para-lacto-N-neohexaose (pLNnH) and para-lacto-N-hexaose (pLNH), using Stepwise One-Pot Multienzyme (StOPMe) with in situ-generation of sugar nucleotides in combination of a glycosyltransferase substrate engineering strategy. I successfully synthesized 56 Cbz-tagged HMOs with yields ranging from 69% to 94%. Native HMOs with a free reducing end were obtained by catalytic hydrogenation followed by spontaneous hydrolysis. The chemoenzymatic synthetic process is readily scalable for large-scale production of HMOs in the future. The products are essential probes and reagents for elucidating roles of HMOs and exploring their applications. HMO-protein neoglycoconjugates with controlled glycan valencies were synthesized and used for binding assays. Six plant lectins were tested in the binding assay and results were consistent with those reported previously. In future studies, additional glycan-binding proteins of interest will be investigated.